Project Summary Alcohol abuse remains one of the leading causes of preventable death13. Yet, a number of factors including stress2,3 and a history of alcohol use17 trigger the initiation or resumption of severe problem drinking making clinical management of alcohol abuse disorder (AUD) difficult. For this reason, a broader range of pharmacotherapies for alcohol abuse disorder (AUD) are needed. Due to the growing interest in compounds that target the serotonin 2A receptor (5-HT2AR) subtype to treat AUD4,58 and the poorly-understood neural substrates mediating these therapeutic effects, this proposal seeks to better understand the mechanism linking 5-HT2AR stimulation with positive treatment outcomes for alcohol abuse. To address this problem, we draw on our recent work in rats where we demonstrated that stress hormone signaling in the VTA precipitates increased alcohol self-administration via a depolarized GABAA reversal potential (EGABAA) in VTA GABA neurons9. The molecular linchpin for this interaction between stress and alcohol is the potassium-chloride cotransporter 2 (KCC2), which was revealed to have diminished chloride transport function in VTA GABA neurons after stress, with similar results observed in mice. Correcting KCC2 function in the VTA was sufficient to reduce stress-related increased alcohol self-administration to the control level. This suggests that drugs with the capacity to upregulate KCC2 may serve as novel AUD pharmacotherapies. Interestingly, recent evidence demonstrates that 5-HT2AR activation in the central nervous system is sufficient to upregulate the function of KCC2 in conditions of impaired chloride transport12. For these reasons, this proposal aims to test the hypothesis that 5-HT2AR activation is sufficient to reduce increased alcohol drinking behavior resulting from stress and chronic alcohol consumption via upregulation of KCC2 in VTA GABA neurons. In Aim 1, stress-induced increases in initiation of alcohol drinking behavior in the intermittent two-bottle choice paradigm will be probed with the 5-HT2AR agonist, TCB- 2. We will determine whether stimulation of these receptors within the VTA can attenuate the onset of alcohol consumption following stress. Aim 2 will investigate the specific mechanism by which 5-HT2AR agonism can reduce alcohol drinking enhanced by stress. Here, the role of 5-HT2AR activation in regulating inhibitory neurotransmission in the VTA will be tested using both indirect (electrophysiology) and direct (western blot) measures to assay KCC2 function. Finally, Aim 3 will relate the results of Aims 1 and 2 to general alcohol abuse. In Aim 3, the intermittent two-bottle choice paradigm will be used to induce high levels of alcohol consumption in mice followed by intra-VTA treatment with TCB-2 to assess the effects of 5-HT2AR activation on chronic alcohol consumption. In parallel, the VTA from separate groups of drinking mice will be studied using both electrophysiology and western blot to examine the potential ameliorating effects of TCB-2 on impaired KCC2 function resulting from chronic alcohol consumption. Taken together, this proposal will provide a novel mechanism connecting 5-HT2AR activation with treatment of AUD.